Download Early-life stress origins of gastrointestinal disease: animal models

Am J Physiol Gastrointest Liver Physiol 309: G927–G941, 2015.
First published October 8, 2015; doi:10.1152/ajpgi.00206.2015.
Review
Early-life stress origins of gastrointestinal disease: animal models, intestinal
pathophysiology, and translational implications
Calvin S. Pohl,1,2* Julia E. Medland,3* and Adam J. Moeser1,2
1
Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, Michigan; 2Gastrointestinal Stress
Biology Laboratory, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan; and 3Comparative
Biomedical Sciences Program, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina
Submitted 25 June 2015; accepted in final form 1 October 2015
irritable bowel syndrome; models; murine; porcine; stress
EARLY-LIFE ADVERSITY
is a major risk factor for the adult onset
and severity of important gastrointestinal (GI) diseases, including irritable bowel syndrome (IBS) and inflammatory bowel
disease (IBD), in adulthood (2, 31, 67, 165). Despite the
established link between early-life stress and GI disease later in
life, the pathophysiological mechanisms remain poorly understood. A number of animal models of early-life stress/adversity
have been employed to study the GI pathophysiology and
health outcomes associated with early-life stress. This review
presents the major GI pathophysiological and clinical findings
in established rodent models and newer large-animal models of
early-life stress. A primary focus of this review is on how the
pathophysiology and clinical findings from animal models of
early-life stress translate to specific human GI disease conditions. Species- and model-related factors that likely play significant roles in predicting the translational value of the models
to humans are emphasized. In addition, this review provides
insight into the relevant postnatal GI developmental tracks that
are altered by a stressful early-life environment, ultimately
leading to a new trajectory toward GI dysfunction and disease
susceptibility later in life.
Stress: Definition and Role and Development of the
Hypothalamic-Pituitary-Adrenal Axis
Definition. “Stress is defined as a state in which homeostasis
is actually threatened or perceived to be so [and] homeostasis
* C. S. Pohl and J. E. Medland contributed equally to this work.
Address for reprint requests and other correspondence: A. J. Moeser, Dept.
of Large Animal Clinical Sciences, Michigan State Univ., East Lansing, MI
48824 (e-mail: [email protected]).
http://www.ajpgi.org
is re-established by a complex repertoire of behavioral and
physiological adaptive responses of the organism” (48). The
neonate and infant are exposed to tremendous, stressful
changes in homeostasis at birth and weaning, and both immunological and hypothalamic system adaptation and plasticity
are necessary for survival. Importantly, the outcome of reestablished homeostasis has major implications for long-term
health. The organism returns to original homeostasis (or eustasis), or the adaptive response creates a new homeostasis. The
new homeostatic parameters can be inappropriate (allostasis)
or beneficial (hyperstasis) (48). Adaptive responses to these
early-life challenges likely dictate health later in life (88) and
are central to long-term GI disease susceptibility. A number of
early-life stressors, including psychosocial (maternal deprivation, loss of caregiver, and physical and emotional abuse) and
immunologic (allergy, infectious, or metabolic/nutritional)
stress, have been implicated as risk factors of GI disease onset
later in life. While these stressors are diverse, they fit the
definition of stress, in that they threaten the host’s homeostasis.
If these stressors occur during a time of significant developmental plasticity, it is likely that new adaptations can reshape
brain and gut function for the individual’s lifetime.
Role and development of the hypothalamic-pituitary-adrenal
axis. The hypothalamic-pituitary-adrenal (HPA) axis is central
to controlling homeostatic adaptations to stress. Psychological
or physical stressors, such as inflammation, are integrated in
the central nervous system (CNS), resulting in a cascade of
positive- and negative-feedback responses mediated by hypothalamic and pituitary release of the stress hormones corticotropin-releasing factor (CRF) and adrenocorticotropic hormone
(ACTH), respectively. ACTH stimulates the cortical adrenal
gland to release glucocorticoids, which have many downstream
0193-1857/15 Copyright © 2015 the American Physiological Society
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Pohl CS, Medland JE, Moeser AJ. Early-life stress origins of gastrointestinal
disease: animal models, intestinal pathophysiology, and translational implications.
Am J Physiol Gastrointest Liver Physiol 309: G927–G941, 2015. First published
October 8, 2015; doi:10.1152/ajpgi.00206.2015.—Early-life stress and adversity
are major risk factors in the onset and severity of gastrointestinal (GI) disease in
humans later in life. The mechanisms by which early-life stress leads to increased
GI disease susceptibility in adult life remain poorly understood. Animal models of
early-life stress have provided a foundation from which to gain a more fundamental
understanding of this important GI disease paradigm. This review focuses on
animal models of early-life stress-induced GI disease, with a specific emphasis on
translational aspects of each model to specific human GI disease states. Early
postnatal development of major GI systems and the consequences of stress on their
development are discussed in detail. Relevant translational differences between
species and models are highlighted.
Review
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ANIMAL MODELS OF EARLY-LIFE GI STRESS
(73, 178). Concurrent with development of the HPA axis early
in life, the gut undergoes a similar extensive maturation period.
Given the bidirectional communications between the HPA axis
and the gut, aberrations in gut development due to environmental stress may directly induce HPA axis dysfunction. Conversely, HPA axis allostasis may contribute to inappropriate
development of the gut. Interruptions in the development of
either system are well associated with risk of developing GI
disease.
Developmental Biology of the Postnatal Intestine
To understand how early-life stress might influence longterm GI development and disease susceptibility, it is important
to consider the major intestinal developmental changes and
adaptations across models of different animal species in the
immediate postnatal period. In all mammals, the early postnatal
period is marked by major developmental changes in preparation for long-term survival. Some major systems that undergo
extensive development and programming during this time are
the enteric, immune, and nervous systems, epithelial barrier
function, and microbiota colonization and composition (124).
While the early-life developmental changes in these systems
allow the host to survive and thrive in the extrauterine environment, perturbations in normal developmental processes by
stress during these periods of plasticity can lead to a deviation
in long-term function of the GI system and an increase in
disease susceptibility (Fig. 1).
Fig. 1. Proposed paradigm of early-life stress and gastrointestinal (GI) disease development. Evidence from rodent and porcine models and human data
demonstrate that early-life stress is a major risk factor in GI disease development and severity later in life. Early-life psychosocial stressors occur during high
developmental plasticity (green) and initiate a trajectory toward increased GI disease susceptibility (red line) later in life. Animal models, such as neonatal
maternal separation (NMS) and colonic irritation in rodents, maternal separation (MS) in nonhuman primates, and early-weaning stress (EWS) in pigs, can be
used in the study of early-life stressors at times of intestinal development (comparable to human perinatal and childhood intestinal development) and enhanced
susceptibility to development of GI dysfunction later in life (adulthood). Common mechanisms of early-life stress-induced disease between animal models (boxes
at right) are increased intestinal permeability, altered microbiota, increased enteric nervous system activity, heightened mast cell numbers and activation,
corticotropin-releasing factor (CRF), cholinergic nervous system [choline acetyltransferase (ChAT)], substance P (SP), and serotonin (5HT). Collectively, these
mechanisms can result in clinical signs of GI disease, including abdominal pain, diarrhea, constipation, and increased susceptibility to enteric infections.
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physiological effects that help the host quickly adjust to the
environmental changes that initially induced hypothalamic
release of CRF (48, 49).
During infancy, childhood, and early adolescence, the HPA
axis is mostly hyporesponsive, with low basal glucocorticoid
secretion until near puberty (106, 112, 151, 180). In humans
and rodents, severe environmental stressors in the absence of
parental care disrupt HPA axis maturation, and these early-life
stressors, characterized by inappropriate release of stress hormones such as cortisol during subsequent stressful conditions,
are associated with development of adult psychological and GI
disease, such as IBS (63, 75, 166, 178). During the neonatal
stage, the adrenal gland is highly sensitive to small quantities
of ACTH, while the negative glucocorticoid receptor-feedback
system is poorly developed (55, 177); thus HPA axis activation
induced by early-life stress results in high and prolonged
glucocorticoid production. Elevated glucocorticoids are detrimental to HPA axis and glucocorticoid receptor development;
thus, prepubescent individuals exposed to early-life stress exhibit exaggerated CNS/behavioral and physiological responses
to subsequent stressful episodes later in life (3, 44, 119). These
phenomena support the hypothesis that early-life stress stimulates the HPA axis during a period when the neuroendocrine
system is intended to be hyporesponsive. Inappropriate stimulation of the HPA axis during the intended hyporesponsive
period results in altered development and life-long allostasis.
In health and disease, the HPA axis is known to play a role
in regulation of gut function; thus the HPA axis has been
established as an important component of the brain-gut axis
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ANIMAL MODELS OF EARLY-LIFE GI STRESS
genic, immune system. Exogenously, maternal milk provides
immune-supportive factors such as secretory IgA, maternal
leukocytes, and milk glycans, all of which modulate and
neutralize intestinal microbes. Additionally, breast milk provides massive amounts of anti-inflammatory cytokines and
peptides, which negatively regulate neonatal Toll-like receptor
and inflammatory cytokine expression (134). Endogenously,
several pathways are involved in inhibition of the innate
immune system, which, in turn, leads to polarization toward a
tolerogenic lymphocyte population early in life (14, 52, 65,
76). However, the neonatal immune system is not inherently
unresponsive or defective. Presence of the commensal microbes induces neonatal immune activity, represented by development of secondary lymphoid organs in mice, pigs, and
humans during the postnatal period (14, 19, 150). Clinically,
neonates can respond to vaccination, although the response is
weak. Additionally, various types of leukocytes from the neonate can be stimulated to induce inflammation. Finally, in early
and abruptly weaned pigs, antibodies to new feedstuffs can be
detected (14, 52, 65). These observations highlight the inflammatory capability of the neonate and reinforce the idea that
there may be consequences to immune overstimulation during
this quiescent period.
At weaning, the immunosuppressive dominance gives way
to a spike in inflammation. Mast cell degranulation and proliferation, intraepithelial lymphocyte proliferation, mucosal inflammatory cytokine induction, and T-cell stimulation coordinate the homeostatic adjustment to weaning (52, 142). Host
inflammatory and metabolic pathways are also upregulated
with weaning to cope with a dynamic microbiota and an
introduction to novel food antigens, factors that are likely
controlled by Toll-like receptor and IL-1 pathways (29, 147).
Weaning can be abrupt or gradual, can occur at different stages
of development, and is associated with an inflammatory response; thus the time and/or developmental stage at which
weaning occurs can interrupt the tolerogenic period. This
becomes particularly important in stressful situations such as
early weaning, when premature immune stimulation during the
tolerogenic window of opportunity can have serious health
implications later in life.
Postnatal development of the intestinal epithelial barrier.
The intestinal epithelium undergoes rapid maturation during
the postnatal period. While some postnatal epithelial changes
are thought to be genetically “hard-wired,” many are driven by
environmental, microbial, and endocrine cues. One of the
earliest and most critical epithelial changes in the postnatal
period is establishment of intestinal epithelial barrier function.
Intestinal epithelial barrier function refers to the ability of the
epithelium to form a selectively permeable barrier, regulated
predominantly by tight junction proteins and mucus, which
prevent the vast amounts of luminal antigens, pathogens, and
toxins from gaining entry into the underlying tissues and
systemic circulation. Impairment of the epithelial barrier results in exposure of luminal constituents to the underlying
immune, circulatory, and nervous systems, inciting local neuroinflammatory events and systemic inflammation. Disturbance of this epithelial barrier, characterized by heightened
intestinal permeability, or “leaky gut,” is a hallmark in the
pathogenesis of major GI diseases, including IBD, IBS, celiac
disease, and food allergy/intolerance (38, 100, 128). The postnatal development of intestinal barrier properties has been
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Postnatal development of the enteric nervous system. The
enteric nervous system (ENS) exerts regulatory control over
numerous GI functions, including motility, visceral sensation,
secretion, and absorption, and immune and epithelial barrier
function (77, 79, 122). Therefore, alterations in ENS function
can lead to profound clinical GI symptoms, which represent a
central pathophysiological process in stress-related GI disorders. Although the ENS can operate independently of CNS
input, the ENS is essential in integration of signaling between
the gut, the CNS, and the brain. Major developmental processes and adaptations that exhibit a high degree of plasticity
take place in the ENS during postnatal life. Given the plasticity
of this system, stressful or harmful stimuli in early postnatal
ENS development have the potential to alter normal ENS
development and life-long function. Key ENS postnatal processes include the formation of functional neurocircuits, gangliogenesis, differentiation of neuron phenotypes, and neuron
cell death (111, 154). After neurogenesis and gangliogenesis,
the ENS undergoes a normal decline in the number of neurons
via apoptosis, as demonstrated in laboratory animals and humans (7, 80, 155, 186). Although the mechanisms of “ENS
pruning” in the postnatal gut are incompletely defined, it is
thought that these mechanisms might include loss of appropriate survival factors (36, 41), possibly including nerve growth
factor (NGF) and glial-derived neurotrophic factor (74, 109,
179, 182). In addition, throughout the postnatal period, the
neurochemical composition of the ENS changes significantly.
Of particular importance are alterations in cholinergic innervation to the gut, where the proportion of neurons expressing
acetylcholine, the major excitatory neurotransmitter in the GI
tract, increases dramatically (58), often doubling and accounting for ⬃44% of all neurons in the submucosal plexus and 62%
of all neurons in the myenteric plexus by maturity (78, 93).
ENS neurite outgrowth is another important postnatal event
(77). In summary, the ENS undergoes significant development
and maturation during the early postnatal period and exhibits a
high degree of plasticity. Therefore, an understanding of how
early-life stress influences normal ENS development could be
critical to the understanding of early-life stress-induced GI
disease.
Postnatal development of the GI immune system. In childhood, birth and weaning represent major challenges for earlylife host immunity. At birth, the host must adapt to microbial
colonization of the lungs, intestine, and skin, as well as consumption of milk antigen, all of which have the potential to
induce massive inflammation. Similarly, at weaning, the host
must cope with the psychological stress of maternal separation
(MS) or deprivation while also adapting to a sudden exposure to food antigens and changes in microbial community
without the support of maternal immunity. Massive change
in the gut transcriptional profile at birth and weaning indicates the adaptive effort of the host during disruptions in
homeostasis (147). Ontologically, the early-life immune system has been described as suppressed, yet active (52, 76). It is
hypothesized that the perinatal period is a “window of opportunity” for tolerogenic induction and that excessive inflammatory interruption during this period may lead to maladaptive
responses with long-term health consequences (14, 150). Research over the last 20 years has described multiple layers of
exogenous and endogenous immunosuppressive mediators that
modulate infant immunology to promote an active, yet tolero-
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milk to solid food at weaning and may play a role in diseases
such as IBD (6, 54, 188). One of the key roles of the microbiota
in the neonate is establishment of oral tolerance to commensal
microorganisms and food (104, 162). Additional roles of the
microbiota in the developing mucosal immune system include
development of gut-associated lymphoid tissue and intestinal
lymphocytes and antimicrobial peptide secretion into the lumen (102). Neonatal colonization is also required for normal
neurological development, including development of the HPA
axis (62, 133, 161), which further highlights this period as a
critical window in development.
In summary, tremendous and complex developmental
changes occur in the GI tract during early postnatal life. During
this time, enteric neuronal, immune, epithelial, and microbial
signaling act in concert to prepare the host for adaptation to,
and survival during, the immediate and long-term postnatal
environment. In addition, the enteric systems exhibit a high
degree of plasticity; thus disturbances in the normal developmental windows, such as early-life stress/adversity, can lead to
long-lasting changes in intestinal function and disease susceptibility.
Animal Models of Early-Life Stress-Induced GI Disease
Several animal models have been employed to investigate
the impact and mechanisms of early-life stress on GI disease.
While all these models (see below) are centered on disruption
of the early-life environment with psychosocial stress or chemical injury, they differ in terms of stressor types, species
variations, and clinical and pathophysiological outcomes. We
describe the established animal models of early-life stress used
for GI disease investigations, with a focus on model and
species differences, pathophysiological findings from each
model, and translatability to human GI disease.
Models of Early-Life Stress-Induced GI Disease in Rodents
Neonatal MS. The most commonly utilized animal model of
early-life stress is the neonatal MS (NMS) model in rodents.
There are several different types of NMS, including short
handling, where the pups are handled for 15 min/day during the
postnatal period, and long MS, where the pups are separated
from the dam for 3 h/day. Here we focus mainly on the
long-NMS model, as few studies have used the short-NMS
model to address GI outcomes. Nonetheless, unlike long NMS,
short NMS (handling) has been shown to be protective (decreased anxiety responses) (120, 125) and does not result in
long-term GI dysfunction (136). In long NMS, rat or mouse
pups are separated from their dam daily for 3-h periods: rat
pups between 2 and 14 days of age or between 4 and 20 days
of age (25, 84) and mouse pups between 1 and 14 days of age
or between 1 and 18 days of age (12, 114). While the majority
of NMS research has been carried out in mice, much of the
NMS research concerning GI function has been performed in
rats. The NMS model is based on disruption of development of
the HPA axis by induction of stress and HPA axis activation
during the hyporesponsive period (4 –14 days of age in rodents) (135, 143), as discussed previously in this review.
Among the most-studied GI-related effects of NMS in rodents
are long-term changes in GI motor (e.g., motility) and sensory
(e.g., visceral hypersensitivity) function. Adult rodents that
were exposed to NMS exhibited delayed gastric emptying and
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investigated in multiple animal species and humans. At birth,
the neonatal intestinal barrier is highly permeable; it matures
during the postnatal period, as indicated by a progressive
decline in permeability with age; however, species-specific
variations exist. In full-term human infants, Weaver et al. (183)
showed that GI permeability (measured as urinary lactuloseto-mannitol ratios) remained stable during the first 4 days of
postnatal life. Catassi et al. (40) demonstrated that GI permeability, also measured as urinary lactulose-to-mannitol ratio,
declined significantly, by ⬃3.7-fold, between 1 and 7 days of
age, indicating a rapid postnatal decline in GI permeability
(40). They also showed that breast feeding accelerated the
decline in GI permeability (40). GI permeability was markedly
higher in preterm than full-term infants and then declined
rapidly with age (175, 183). In mice, GI permeability (measured by in vivo FITC-4-kDa dextran permeability) is high at
birth and declines with age; however, the most pronounced
reductions in GI permeability occur later in mice (at 2–3 wk of
age) than in humans (139). Similarly, in neonatal rabbits, small
intestine permeability was high at birth and then declined
progressively into adulthood (⬎120 days) (171). In full-term
piglets, GI permeability [measured by in vivo lactulose-tomannitol ratio (sugar absorption tests)] remained stable after
birth, with little change between birth and 10 days of age (98).
However, utilizing ex vivo jejunal preparations on Ussing
chambers, De Quelen et al. (56) reported that intestinal permeability increased between 0 and 14 days of age and declined
thereafter. Together, these findings indicate that while many
species exhibit a postnatal maturational decline in intestinal
permeability, the time course is very different. The implications for these species and, therefore, model differences relative to human clinical relevance are discussed later in this
review. In addition to the developmental aspects of intestinal
epithelial permeability, other key barrier and innate epithelial
cell changes, including marked changes in the expression and
repertoire of antimicrobial peptides, pattern recognition receptors, and immune signaling pathways (144), nutrient transporters (110, 168), and crypt-epithelial regenerative complexes
(57, 94), also occur in the postnatal period. Furthermore,
similar to the other GI system changes described above, postnatal epithelial development is modulated and shaped extensively by dietary, microbial, neuroendocrine, and environmental influences and differs by species.
Postnatal establishment of the enteric microbiota. The microbiota exerts a large influence on GI function and health
throughout life, but its composition is determined largely
during the postnatal period (71, 90, 132). While most of the
available literature suggests that colonization of the GI tract
occurs at birth, with first exposure in the vaginal canal (90,
181), there is also evidence to suggest that colonization occurs
in utero (101). Given that this is the founding group of bacteria,
any abnormal stress or inflammatory state of the mother can
influence the microbiota of the offspring (141, 181). Breast
milk has a profound impact on the microbiota and further aids
in colonization, with a higher proportion of Bifidobacteria in
breast-fed than formula-fed individuals (71, 181, 189). At
weaning, the microbiota is subject to great change with the
transition from breast milk to a solid diet (71, 147, 148), and
this transition coincides with a period of gut maturation (148).
The effect of diet (e.g., high fat and carbohydrate availability)
on the microbiota continues after the transition from breast
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ANIMAL MODELS OF EARLY-LIFE GI STRESS
are consistent with those reported by Dothel et al. (64), who
showed increased nerve fiber outgrowth and NGF in adult
human IBS biopsies.
Another consistent GI pathology observed in the NMS
model is persistent elevation of GI permeability. Numerous
investigations utilizing the NMS model demonstrated transcellular and/or paracellular permeability defects in adult rats that
had been subjected to NMS (22, 84, 85, 114, 159). Increased
intestinal permeability in this model is highly relevant to
human GI disorders, as increased intestinal permeability is a
well-established pathophysiology in diseases including IBS,
IBD, and food allergy (39) and is linked with abdominal pain,
inflammation, and disease susceptibility. The mechanism by
which early-life stress induces persistent defects in intestinal
permeability in humans and animals remains to be fully elucidated; however, studies utilizing the NMS model have shown
that activation of multiple signaling pathways is involved. CRF
receptor signaling pathways have been shown to mediate
NMS-induced intestinal permeability, as peripheral administration of CRF receptor antagonists reduced intestinal permeability in NMS rats (84, 85, 159). In addition, as described
previously, enteric muscarinic receptor blockade also inhibited
intestinal permeability in NMS rats, suggesting an interplay
between enteric cholinergic nerves and CRF receptors in regulating NMS-induced intestinal permeability.
There have been several investigations of the impact of
NMS on subsequent GI inflammatory and/or psychological
stress responses later in life. Barreau et al. (21) showed that rats
exposed to NMS were more susceptible to infection by Nippostrongylus brasiliensis. In another study, Barreau et al. (22)
demonstrated increased colonic myeloperoxidase, numbers of
mast cells, and expression of cytokines in rats that had been
subjected to NMS. Furthermore, administration of 2,4,6-trinitrobenzenesulfonic acid induced higher inflammatory and intestinal permeability responses in NMS rats than in normalreared controls (22). Similarly, adult rats and mice that had
been subjected to NMS exhibited a worsening of colitis induced by dextran sulfate sodium (129, 176). Lennon et al.
(114) demonstrated that while NMS had no effects on colonic
cytokine levels and colitis histological scores in adult wild-type
mice, adult IL-10⫺/⫺ mice exposed to NMS exhibited an early
onset and increased severity of colonic cytokine levels (increased IL-12 p40 and IFN-␥ mRNA) and colitis scores and
intestinal permeability. They also showed that NMS and IL-10
deficiency contributed to increased intestinal permeability in
the absence of marked inflammation, suggesting that both
IL-10 deficiency and NMS were required to induce the onset of
severe colitis. This is consistent with a “two-hit” (NMS and
IL-10 deficiency) theory that has been associated with human
IBD, as well as other GI diseases. Findings from the NMS
model and colitis susceptibility are consistent with human
evidence of early-life adversity leading to increased chance of
developing IBD in humans (1, 2, 70). MS animals also show
greater reactivity to psychological stressors, such as water
avoidance or restraint stress, later in life. In response to these
acute stressors, animals subjected to NMS display increased
visceral hypersensitivity (27, 51, 152, 156, 172, 174), increased
intestinal short-circuit current (Isc) and macromolecular permeability (159), increased numbers of mast cells (172), increased
motility (27, 156), and increased fecal output (51). While
human data for responses of IBS patients to acute stressors are
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accelerated colonic transit (13, 33). Increased fecal pellet
output (an indirect measure of increased motility) was observed in NMS rats that were subjected to psychological
stressors later in life (27, 51, 156), indicating heightened or
exaggerated motility responses to adversity. Fecal output and
altered colonic transit, while not identical, are comparable to
symptoms of altered bowel frequency, such as diarrhea or
constipation, in IBS in humans (66, 69). The effects of NMS on
visceral sensitivity, a surrogate marker for abdominal pain,
have also been well documented. Because abdominal pain is a
key symptom of stress-related GI disorders such as IBS (4),
study of the effects of NMS on visceral sensation is highly
relevant to human GI disorders. A number of investigations
(50, 51, 60, 89, 152) have demonstrated lasting visceral hypersensitivity, as determined by increased sensitivity (lower
threshold) to colonic distension, in rats subjected to NMS.
There have been several reports of the differences between
males and females exposed to NMS, and the most prominent
differences were in HPA function, where females appear to
have a more reactive HPA response (61). Rosztoczy et al. (152)
demonstrated greater visceral hypersensitivity in females than
males in response to two MS protocols: removal of all pups
from their home cage or separation of half of the pups from
their littermates. The increased visceral hypersensitivity in
female rodents is consistent with the paradigm of human IBS,
where disease is more prevalent in females. However, there is
a paucity of data concerning the mechanistic differences in the
GI system between male and female rats exposed to NMS.
Further investigations into the mechanisms of NMS-induced
visceral hypersensitivity in rodents showed that mast cell
degranulation, CRF receptor activation, transient receptor potential cation channel (TRP) subfamily V member 1, and
increased intestinal permeability were central mechanisms in
the visceral hypersensitivity observed in this model (156, 172).
These mechanisms appear to be similar to those proposed in
human IBS pathophysiology; thus, mast cells, CRF receptors,
and voltage-gated sodium channels are currently targets for
drug development for human GI disorders associated with
abdominal pain (4, 44).
In addition to motility and visceral hypersensitivity changes
induced by NMS, changes in neurochemical phenotype of
enteric neurons have been reported. Gareau et al. (84) demonstrated that NMS resulted in increased numbers of cholinergic
(choline acetyltransferase-positive) enteric nerves in 20-dayold weanling pups. They also reported that ex vivo application
of the muscarinic receptor antagonist atropine reduced the
elevated permeability of intestinal tissues from NMS rats (84),
indicating a functional role of cholinergic signaling in NMSinduced permeability changes. The role of cholinergic function
in the early-life stress GI disorders has not been further
investigated in rodents but could be a major target in human GI
disease. Serotonin, a major neurotransmitter involved in motility, secretion, and visceral hypersensitivity, was elevated in
the colon of NMS rats following water avoidance stress or
colonic distension (27, 149). Increased serotonergic signaling
in the NMS model has important implications in human IBS, in
which dysregulated serotonin signaling is thought to play a
significant role in GI symptoms (37). NMS rats were also
shown to exhibit increased intestinal mucosal nerve fiber density and synaptogenesis in the GI tract, which was prevented by
administration of an antibody against NGF (25). These findings
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rats displayed decreased lumbosacral activation thresholds and
increased amplitude of lumbosacral response (117).
Models of Early-Life Stress-Induced GI Disease in Large
Mammals
Porcine early-weaning stress model. A large-animal model
that has emerged as a valid model to study early-life stressinduced GI disease is the porcine early-weaning stress (EWS)
model. The EWS model is based on interruption of the natural
weaning process, with an abrupt and stressful early weaning.
Weaning in mammals is defined as the transition from maternal
breast milk to solid food or infant formula (96). In nature,
weaning is a prolonged, gradual process as the nursing animal
or infant transitions to food and social independence. In the
EWS model, piglets are removed from their dam at an early
age (15–18 days of age) compared with a gradual weaning over
3 mo, as in nature (14). Weaned piglets are moved to a nursery
environment that encompasses a number of additional stressors, including psychosocial (e.g., maternal and sibling separation, transport and transition to a new environment, fighting,
and establishment of a new social hierarchy) and immunological (e.g., exposure to new dietary antigens and pathogens)
stressors. Moreover, animals are exposed to weaning stressors
during a period of significant postnatal GI development, as
mentioned previously. Given the complex stressful events
associated with early weaning, EWS provides a novel model of
adverse early-life events, such as loss of caregiver, abandonment, or early, abrupt transition to formula or solid food.
Furthermore, early weaning is a routine practice in animal
agriculture systems; therefore, studying the mechanisms of
EWS provides an opportunity to understand disease mechanisms and improve the health and well-being of agricultural
animals.
Under normal, unstressed housing conditions, EWS pigs are
generally healthy and exhibit growth rates similar to those of
late-weaned (weaning age ⬎23 days) control pigs. In response
to weaning, EWS and late-weaned control pigs exhibit marked
elevations in serum CRF and cortisol (131), indicating that,
regardless of the age of the animals at weaning, the perceived
stress is comparable. However, baseline GI pathophysiology
and stress reactivity are markedly different between EWS pigs
and controls. EWS pigs exhibit a chronic, relapsing functional
diarrhea that persists into adulthood (unpublished observations). Compared with rodent models, the chronic relapsing
diarrhea is a unique clinical feature in the porcine EWS model
and is relevant and translatable to human chronic-stress diarrheal conditions such as diarrhea-predominant IBS. When
faced with a later-life enteric pathogenic challenge (enterotoxigenic E. coli), EWS pigs exhibit a more rapid onset and
severity of diarrhea (126). In comparison, in humans, early or
premature (⬍4 – 6 mo of age) weaning in infants was shown to
be a risk factor for developing subsequent gastroenteritis (68,
113). Therefore, the EWS pig could be a valid model of
specific weaning-related GI disorders in humans. The mechanisms of diarrhea that are observed in the EWS model are not
completely understood but are likely due to heightened secretory mechanisms. Increased intestinal Isc, a measure of net
electrogenic ion transport, has been observed in the EWS pig
intestine compared with unweaned or late-weaned controls
(130, 158). The increased Isc tone in the EWS intestine was
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limited, increased cortisol, GI symptoms, and skin conductance
in response to an acute stress test have been shown (105).
Together, these studies show that NMS predisposes rodents to
inflammatory and psychological stressors, leading to exacerbated intestinal injury.
As described previously, the microbiota undergoes significant development in early in life and has emerged as a key
player in a number of GI diseases in humans. A few investigations have shown that NMS in rodents alters the composition
of the microbiota (20, 81, 83). Specifically, decreased populations of Lactobacillus species (83) and increased total numbers
of adherent bacteria (83), with specific increases in Clostridia,
Enterococcus, and Escherichia coli species (20), have been
reported. Additionally, administration of probiotics reduced
transcellular intestinal permeability and ion transport in NMS
rodents (83).
Limited-nesting stress. In the limited-nesting stress model of
early-life stress in rats or mice, the dam is given insufficient
nesting/bedding material on postnatal days 2–9 (92, 145). The
goal of this model is to disrupt normal maternal care, thus
mimicking child abuse and neglect (89). Relatively few studies
have measured GI-related outcomes with this model; however,
pups exposed to limited nesting display visceral hypersensitivity (92, 145). Therefore, the limited-nesting stress model is
relevant to GI disease-associated early-life stress and abdominal pain (e.g., IBS) (89).
Odor-attachment learning. Odor-attachment learning is a
model of early-life stress specifically designed to mimic an
abusive caregiver (89). At 8 –12 days of age, rat pups are
conditioned with different types of odor-conditioning: paired
conditioning, where an electric shock is paired with a final odor
stimulus, and unpaired conditioning, where an electric shock
occurs 2 min after the final odor stimulus, which resulted in no
association between the shock and the odor (42, 170). Unpaired
odor shock results in long-term visceral hypersensitivity (170),
which is particularly pronounced in females (42). Therefore,
odor conditioning is a valid model for studying the effects of
unpredictable neonatal stressors on long-term GI function and
pain.
Neonatal colonic inflammation. Neonatal colonic irritation
models involve administration of a chemical irritant or antigen
(e.g., LPS, mustard oil, or acetic acid) to the animal at ⬃5–10
days of age (89). These protocols inflict significant injury to the
colon and are particularly effective for modeling the effects of
neonatal GI infection and trauma. Several neonatal colonic
inflammation studies reported induction of visceral hypersensitivity (5, 47, 187). Additionally, neonatal colonic inflammation and visceral hypersensitivity appear to be associated with
TRP subfamily V member 1 (187) and TRP subfamily A
member 1 (47). Exposure of neonates to LPS resulted in
increased mast cell degranulation and circulating IL-1␤ in
response to a subsequent irritant (formalin) later in life (191).
Neonatal repeated colonic distension. Repeated colonic distension, another model of neonatal GI injury, is typically
performed at 8, 10, and 12 days of age in rat pups (5, 43, 116,
117). Similar to neonatal colonic inflammation, repeated colorectal distension also produces visceral hypersensitivity (5).
Neonatal exposure to repeated mechanical irritation of the
colon has been reported to result in increased stool liquidity,
increased mucosal permeability (conductance), and increased
potassium chloride-induced contractions (43). Additionally,
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ANIMAL MODELS OF EARLY-LIFE GI STRESS
EWS model was shown to involve activation of the intestinal
CRF receptor system (130, 131, 137, 158). As with rodent
models of early-life stress and the human GI diseases, the
interplay between the ENS, the CRF system, and mast cells is
clearly evident in the porcine EWS model. Together, these
findings suggest that the ENS-CRF-mast cell axis represents a
conserved pathophysiological response to early-life stress
across different species and different types of early-life stressors. In addition to providing a model of early-life stress/
adversity, the porcine EWS model also provides a model to
study the effects of weaning age and formula in infants on
long-term GI development and health outcomes later in life. In
summary, EWS in pigs and humans involves multiple stressors, including psychological, immunological, and dietary,
which together induce GI injury during a highly developmental
and plastic period in GI development (Fig. 1). Given the
similar pathophysiology and clinical GI outcomes induced by
EWS and early life adversity in pigs and humans, respectively,
the porcine EWS model holds promise as a valuable translational model for human GI disease linked with adversity early
in life.
Models of Early-Life Stress-Induced GI Disease in
Nonhuman Primates
MS in nonhuman primates. The impact of early-life stress
has also been studied in a nonhuman primate model of MS.
Infant primates were peer-reared, separated from their mother
at birth and placed in a peer group at ⬃1 mo of age, or
mother-reared until ⬃7 mo of age (15, 72, 153). While several
behavioral (decreased locomotion and increased stereotypical
behaviors) and neuroendocrine (increased cortisol and serotonin transporter gene expression) changes were noted, few GI
effects were characterized. There is, however, evidence that
MS in rhesus macaques alters the development of the microbiota, with lower numbers of intestinal bacteria, specifically, a
reduction of Lactobacillus species (15). Changes in the microbiota of NMS rodents or MS nonhuman primates are comparable to those observed in IBS and IBD (99, 121).
Translational Considerations for Animal Models of EarlyLife Stress-Induced GI Disease
The early-life stress animal models discussed here, along with
their major GI pathophysiological and clinical outcomes and
strengths and limitations, are presented in Table 1. As demonstrated in Table 1 and discussed in the text, several animal models
of early-life adversity span multiple species. While some common
pathophysiology and mechanisms mimic clinical GI disease states
in humans, there are inherent differences with regard to species
and models that present advantages and/or limitations within the
context of translatability to humans.
Regardless of the early-life stressor used in animal models of
early-life adversity (e.g., NMS, EWS, or colonic irritation), it
is evident that common pathophysiologies, including increased
intestinal permeability, altered ENS development, upregulation
of the CRF system, and mast cell activation (Fig. 1), are
conserved across species. While it is essential that valid animal
models possess mechanisms similar to human disease, the
nature of the stress and comparative genetic and biological
differences across different animal models and species (compared with humans) is significant. Therefore, the species used
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inhibited by pharmacological blockade of CRF receptors, mast
cell degranulation, and enteric nerves (130, 131, 158), suggesting an interplay between the ENS and mast cells in this model.
Compared with the rodent NMS model, EWS in pigs induces both immediate (within 24 h) and long-lasting increases
in intestinal permeability (130, 131, 158). Intestinal permeability in EWS pigs is associated with disruption of the expression and localization of tight junction proteins, including occludin, claudins, and zonula occludens-1 (97, 140). Intestinal
barrier defects in EWS pigs are most pronounced within 24 h
postweaning but persist throughout life. Comparably, human
infants weaned directly onto formula after parturition exhibited
increased GI permeability compared with age-matched, breastfed infants, and this increased permeability persisted for ⱖ1
wk during the postnatal period (40, 184). Additionally, early
weaning onto formula in infants has been correlated with
increased risk for IBD and celiac disease (108, 164). The
mechanisms for increased GI permeability and disease susceptibility in early-weaned infants are not known but could be
similar to mechanisms defined in the porcine EWS model (e.g.,
CRF and mast cell-dependent pathways). Dietary factors (e.g.,
milk protein, fat, and growth factors), immunological stimuli
(e.g., introduction to novel antigen), and psychological factors
likely also play a role in intestinal permeability in EWS pigs
and early-weaned infants. In the pig, diet (milk vs. cerealbased) (30) and dietary soy antigens (115) have been demonstrated to influence intestinal inflammation and function in the
newly weaned pig and, therefore, could also play a role in
intestinal permeability disturbances observed at weaning.
A predominant histopathological feature of the porcine EWS
model is the presence of increased numbers of intestinal mast
cells and their active degranulation status (131, 158). As
mentioned previously, mast cells are critical stress effector
cells within the brain-gut axis and have been shown to be
increased in many stress-related and allergic GI disorders. For
example, IBS is associated with increased numbers of mucosal
mast cells, and activated mast cells have been correlated with
clinical symptoms of abdominal pain and diarrhea in humans
(16 –18). Mast cells are capable of releasing numerous prestored granule mediators (e.g., histamine, TNF, and proteases)
as well as synthesized mediators (e.g., cytokines, chemokines,
and lipid-derived mediators). Released mast cell products can
act on numerous cell types, triggering increases in secretion,
permeability (epithelial and endothelial), immune cell recruitment, and enteric nerve depolarization. The functional significance of heightened mast cell activity in the porcine EWS
model was demonstrated in studies by Smith et al. (158) and
Moeser et al. (131), where intraperitoneal administration of the
mast cell-stabilizing agent sodium cromolyn reduced intestinal
permeability in EWS pigs, demonstrating that persistent mast
cell activation is a central mechanism in intestinal permeability
defects in the EWS model. Interestingly, despite the intestinal
permeability defects and persistent mast cell activation in EWS
pigs, histological lesions and inflammatory responses are limited. In contrast, NMS has been shown to increase baseline
inflammation in rats (22). The lack of significant histological
inflammation in pigs, along with the significant functional and
clinical GI abnormalities, is consistent with biopsy findings in
human IBS. The precise mechanisms by which mast cells are
regulated by EWS remain to be elucidated; however, EWSand mast cell-mediated intestinal permeability in the porcine
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ANIMAL MODELS OF EARLY-LIFE GI STRESS
Table 1. Animal models of early-life stress-induced GI disease
Early-Life Stress Models
in Large Mammals
Early-Life Stress Models in Rodents
Neonatal colonic
injury
Dam is given insufficient
nesting/bedding
material on postnatal
days 2–9
Pups are conditioned
with an odor and
an electric shock
(paired and
unpaired) between
postnatal days 8
and 12
● Anxiety and
depression-like
behaviors
● 1 Fecal pellet
output in
response to laterlife stress
● Depressive behaviors
● Depressive
behaviors
Pups are subjected to
colonic injury in
the form of
repeated colorectal
distension or
chemical exposure
(e.g., mustard oil)
between postnatal
days 5 and 10 or
8 and 12,
respectively
● 1 Stool liquidity
● 1 Baseline
intestinal
permeability
● 1 Intestinal
permeability in
response to laterlife psychosocial
(WAS) stress
● Colonic
hypermotility
● Visceral
hypersensitivity
Limited-nesting Stress
Protocol
Mice or rat pups are
separated from
the dam for 3
h/day during
postnatal days
2–14 or 4–20 in
rats and 1–14 or
1–18 in mice
Clinical signs
Pathophysiology
● Visceral
hypersensitivity
● Visceral
hypersensitivity
● Females display
more severe
visceral
hypersensitivity
● 1 Mast cell
degranulation and
circulating IL-1␤
in response to a
secondary irritant
● 1 Intestinal
permeability
● Visceral
hypersensitivity
● 1 Motility (with
KCl)
● 1 Mast cell
activation
● 2 Lumbosacral
activation
thresholds
● 1 Central and
enteric CRF
system activity
● Microbiome
alterations
Mechanisms
● 1 Bacterial
attachment to
intestinal
epithelium
● 1 Susceptibility
to colitis
● 1 Cholinergic
nervous system
activity
● CRF system
Strengths
● Murine genetic
models (e.g.,
knockout,
knockin,
transgenic lines)
and tools are
readily available
MS macaques
Macaques are peer-reared,
separated from their
mother at birth and
placed in peer group at
⬃1 mo of age, or
mother-reared until ⬃7
mo of age
● Baseline chronic,
relapsing diarrhea
● 2 Locomotion
● 1 Clinical severity
(diarrhea) in response
to later-life infectious
challenge
● 1 Clinical severity
(diarrhea) to later-life
psychosocial stress
● 1 Baseline intestinal
permeability
● 1 Stereotypical
behaviors
● 1 Intestinal
permeability in
response to later-life
psychosocial (mixing)
stress
● 1 Intestinal
permeability in
response to later-life
infectious challenge
● Suppressed innate
immune response to
later-life infectious
challenge
● 1 Mast cell
activation
● Alterations in fecal
microbiome MS
macaques
● 1 ENS activity
● 1 Activation of
peripheral and enteric
CRF system
● CRF system
● Mast cells
● Nerve growth
factor
● Cholinergic
nervous system
● Serotonin
● Well established
and characterized
EWS porcine model
Early-weaned piglets are
removed from the
sow at 15–18 days
age; control animals
are removed at ⬎24
days of age
● Murine genetic
models (e.g.,
knockout, knockin,
transgenic lines) and
tools are readily
available
● Model mimics some
of the major
pathophysiology
related to human
disease (visceral
hypersensitivity)
● Sex-dependent
mechanisms
● Murine genetic
models (e.g.,
knockout,
knockin,
transgenic lines)
and tools are
readily available
● Model mimics
some of the major
pathophysiology
related to human
disease (visceral
hypersensitivity)
● Mast cells
● CRF system
● Spinal TLR-4
● Hyperpolarizationactivated cyclic
nucleotide-gated
● Mast cells
● ENS
● Well-established
model for visceral
hypersensitivity
● Established and
repeatable animal
model
● Good model for
infection and
trauma
● Model mimics some
of the major
pathophysiology
related to human GI
disease
High degree of biological
similarity to humans
Continued
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Odor-attachment
learning
NMS
Early-Life Stress Models
in Nonhuman Primates
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ANIMAL MODELS OF EARLY-LIFE GI STRESS
Table 1.—Continued
Early-Life Stress Models in Rodents
NMS
Limited-nesting Stress
Odor-attachment
learning
● Model mimics
some of the
major
pathophysiology
related to human
disease
References
● Murine genetic
models (e.g.,
Knockout,
knockin,
transgenic lines)
and tools are
readily available
● Model mimics
some of the major
pathophysiology
related to human
disease
● Irrelevant
stressors
compared with
human stressors
● Clinical features
(increased pellet
output) not
directly
comparable to
human GI
symptoms
● Significant
differences in
murine GI
development and
nervous and
immune systems
compared with
humans and large
animals
● Clinical features not
directly comparable to
human GI symptoms
9–11, 22–25, 27, 33,
51, 82, 84, 95,
118, 127, 135,
163, 169, 172,
190
8, 89, 92, 145, 146
● Significant differences
in murine GI
development and
nervous and immune
systems compared
with humans and large
animals
● Relatively little GI
data available outside
visceral
hypersensitivity
Early-Life Stress Models
in Nonhuman Primates
EWS porcine model
MS macaques
● Chronic, relapsing
diarrhea in EWS pigs
is comparable to
human GI symptoms
● High degree of
biological similarity
to human GI system
(e.g., development,
anatomy, immune
system, CNS, and
ENS complexity)
● EWS model has dual
purpose/benefit to
study effects of earlylife stress in
agricultural animal
species
● EWS model could be
used to study effects
of early weaning and
infant formula in
human infants
● Expensive (compared
with rodents)
● Irrelevant
stressors
compared with
human stressors
● Clinical features
not directly
comparable to
human GI
symptoms
● Stressor is very
invasive
● Ethical concerns of
using primates in
research
● Clinical features
not directly
comparable to
human GI
symptoms
● Requires specialized
housing facilities
● Expensive
● Significant
differences in
murine GI
development and
nervous and
immune systems
compared with
humans and large
animals
● Relatively little
GI data available
outside visceral
hypersensitivity
● Significant
differences in
murine GI
development and
nervous and
immune system
compared with
humans and large
animals
● Abdominal pain or
hypersensitivity not
yet investigated
● Requires specialized
housing facilities and
expertise
● A few GI effects and
mechanisms have yet
to be characterized
42, 89, 146, 170
5, 43, 45–47, 116,
117, 187, 191
● Limited genetic
models and reagents
available for pigs;
however, techniques
in this field are
rapidly progressing,
and resources are
becoming more
available
126, 130, 131, 140, 158
15, 72, 107, 153, 157,
160
Features of each model, including methods, clinical features, pathophysiological hallmarks, and advantages and disadvantages, in relation to early-life adversity
resulting in later-life gastrointestinal (GI) disease in humans. CNS, central nervous system; CRF, cotricotropin-releasing factor; ENS, enteric nervous system;
EWS, early-weaning stress; NMS, neonatal maternal separation (MS); TLR, Toll-like receptor.
in the model could have a significant role in the translatability
and eventual therapeutic efficacy in humans. For example,
rodent models employ stressors such as intermittent MS, water
avoidance stress, and restraint stress, which may not be directly
relevant to the complexity of life stressors in humans. In the
EWS model, piglets are subjected to significant psychosocial
trauma as they are removed permanently from their mother and
siblings and forced to adapt to a new environment and social
hierarchy with unfamiliar pigs. Therefore, the design of the
porcine EWS model may more closely resemble human conditions where children are forced to adapt to strenuous conditions without proper parental care.
In addition to the nature of the stress, species differences
(genetic, anatomic, and clinical) present both strengths and
limitations that should be considered in the selection of the
model and the potential translational value. Rodent models
have been, and will likely continue to be, critical to the
understanding of GI diseases associated with stress. Some
obvious strengths of laboratory rodent models are that techniques and reagents for genetic manipulation and molecular
biology are readily available. Several genetic animal models
and approaches (e.g., knockout, knockin, transgenic, and conditional knockdown) are powerful tools to study in vitro and in
vivo contributions of specific mediators. However, genetic
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Limitations
Neonatal colonic
injury
Early-Life Stress Models
in Large Mammals
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ANIMAL MODELS OF EARLY-LIFE GI STRESS
humans and pigs have only 17–18 genes in the granzyme/mast
cell tryptase/serine protease superfamily, while mice have up
to 26 genes in this family (53). Release of these additional and
different proteases in rodents compared with those in humans
and pigs may contribute to different mechanisms of pathology
and disease. Clinically, in the EWS model, pigs develop a
functional, relapsing diarrhea, which has not been reported in
rodents. This unique clinical presentation for an animal model
of early-life stress mirrors stress-related diarrhea in humans
and, thus, can serve as clinical readout, especially when evaluating therapies directed at diarrhea symptoms.
Neurobiological comparison between humans, pigs, and
rodents reveals several differences between species; however,
the HPA axis demonstrates a conserved neurological pathway
for mediating homeostasis between the brain and the gut during
stress across all species. The complexity of the human brain
may be better modeled in the pig, as the human and the pig
possess advanced gyrencephalic neuroanatomy (87). Consistent with their anatomy, several lines of behavioral evidence
indicate that pigs are highly intelligent and capable of performing several cognitive tasks. For example, pigs have been shown
to be able to pass the “mirror test,” indicating that they are
self-aware animals, a property that only primates and a handful
of other mammals are known to possess (86). Therefore, pigs
likely have a more complex cognition of psychological stress
and may have a more complicated brain-gut integration of
stress than rodents.
Finally, inherent differences in diet separate humans and
pigs from mice, because pigs and humans are naturally omnivores, whereas rodents are naturally granivorous. This important distinction explains differences in metabolism and digestive biochemistry between rodents and humans, and these
differences in digestion could influence developmental mechanisms and subsequent clinical outcomes.
Summary
Animal models of early-life stress have provided key insight
into the important brain-gut signaling pathways associated with
long-term changes in GI function and disease susceptibility.
The precise mechanisms linking early-life stress with GI disease remain to be elucidated. Without continued investigations
and development of knowledge in this field, effective therapies
for treating stress-induced diseases associated with early-life
stress will remain limited. Between animal models, many
common biological pathways are activated by early-life stress;
however, inherent species-related differences could have major
translational significance. Rodent models predominate in the
literature and are expected to continue to be extremely valuable
models for study of stress-related GI diseases in humans.
However, large-animal models, such as the porcine EWS
model, are gaining recognition in this field. Given the high
degree of genetic, anatomic, and physiological homology between pigs and humans, large-animal models such as the pig
are expected to offer unique advantages over murine models,
particularly in translation from basic biological mechanisms to
the predictability of therapeutic successes in humans.
ACKNOWLEDGMENTS
The authors acknowledge the expertise of Jessica Hauptman (Michigan
State University) and her help with figure preparation.
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manipulation of large-animal species such as the pig is now
possible and becoming more common. Because of their small
body size, rodent colonies are easy to manage, and the inbreeding of rodent genetic lines reduces animal-to-animal variation
in biological responses. However, human populations are heterogeneous, and variation between individual animals better
reflects human disease conditions and provides a framework
for personalized medicine. On the basis of the EWS studies and
years of research using the pig as a biomedical model, the pig
also is valid model for studying stress-related GI disease in
humans. Moreover, the pig possesses distinct species-specific
translational advantages compared with rodent. It is well accepted that the GI anatomy, physiology, biochemistry, and
evolution are more similar between humans and pigs than
between humans and rodents (32, 91, 103, 185). At full term,
there are common GI developmental pathways shared by all
species, but the neonatal intestine is considered to be in
advanced stages of development in the human and pig compared with rodents. In consideration of histological morphology and epithelial ontogeny, humans and pigs have fully
developed villi and crypts in the small intestine at birth,
whereas the rodent develops intestinal crypts in the postweaning period (59). In addition, as discussed above, the postnatal
development of intestinal barrier function (decline in intestinal
permeability) occurs largely in the first 7 days of life, compared with 14 –21 days of life in mice. Therefore, the morphological ontogeny described above and the delayed decline in
intestinal permeability in mice suggest that, during the first
7–14 days of life, the murine intestine is more comparable to a
preterm human intestine. This could have significant implications for interpreting clinical relevance of murine models of
early-life GI stress/injury that occurs between 1 and 14 days of
age (NMS, colonic neonatal irritation, and limited nesting), as
the GI injury is induced during a relatively underdeveloped
state compared with humans. Ontogeny studies of the ENS
suggest that, at birth, rodents, pigs, and humans have a semimature ENS, which undergoes significant postnatal modification (35, 80, 138, 186). However, the complexity of the human
ENS is more similar to that of the porcine than the rodent
intestine. For example, the human and porcine gut have additional interneuronal networks and plexi that are absent in the
rodents (167). Furthermore, the colocalization of neurotransmitters to certain neuronal subtypes is more common between
humans and pigs than between humans and rodents (32).
Considering the neuronal nature of psychological stress, the
interconnection between the brain and the gut, and the ENS as
a clinical drug target, the added complexity of the porcine ENS
may more similarly mimic human stress-induced GI syndromes and better predict therapeutic efficacy in humans.
Similar to the ENS, the neonatal immune system is present, but
rudimentary, in humans, pigs, and rodents. Considering the
mechanistic role of the immune system in early-life stress, it is
important to note that the frequency of preserved immunologically related genes between humans and pigs is ⬃80%, while
the number of orthologous immunological genes between mice
and humans is only 6% (123). Immunologically, mast cells are
a major component of gut-mediated stress responses and play
a major role in the weaning process in humans, pigs, and
rodents. Mast cell degranulation at weaning is similar across
these three mammalian species; however, the mediators released from mast cells are species-dependent. For example,
Review
ANIMAL MODELS OF EARLY-LIFE GI STRESS
GRANTS
A. J. Moeser acknowledges funding from National Institutes of Health
Grants R01 HD-072968, R03 DK-097462, and K08 DK-097462.
19.
DISCLOSURES
20.
No conflicts of interest, financial or otherwise, are declared by the authors.
21.
AUTHOR CONTRIBUTIONS
C.S.P., J.E.M., and A.J.M. developed the concept and designed the research; C.S.P., J.E.M., and A.J.M. prepared the figures; C.S.P., J.E.M., and
A.J.M. drafted the manuscript; C.S.P., J.E.M., and A.J.M. edited and revised
the manuscript; C.S.P., J.E.M., and A.J.M. approved the final version of the
manuscript.
22.
24.
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